Silva E, Gomes P, Soares-da-Silva P
Institute of Pharmacology and Therapeutics, Faculty of Medicine 4200-319 Porto, Portugal.
Life Sci. 2006 Jul 17;79(8):723-9. doi: 10.1016/j.lfs.2006.02.018. Epub 2006 Mar 6.
The present study evaluated the hypothesis of whether increases in vectorial Na+ transport translate into facilitation of Na+-dependent L-DOPA uptake in cultured renal epithelial tubular cells. Increases in vectorial Na+ transport were obtained in opossum kidney (OK) cells engineered to overexpress Na+-K+-ATPase after transfection of wild type OK cells with the rodent Na+-K+-ATPase alpha1 subunit. The most impressive differences between wild type and transfected OK cells are that the latter overexpressed Na+-K+-ATPase accompanied by an increased activity of the transporter. Non-linear analysis of the saturation curve for l-DOPA uptake revealed a Vmax value (in nmol mg protein/6 min) of 62 and 80 in wild type and transfected cells, respectively. The uptake of a non-saturating concentration (0.25 microM) of [14C]-L-DOPA in OK-WT cells was not affected by Na+ removal, whereas in OK-alpha1 cells accumulation of [14C]-L-DOPA was clearly dependent on the presence of extracellular Na+. When Na+ was replaced by choline, the inhibitory profile of neutral l-amino acids, but not of basic and acidic amino acids, upon [14C]-L-DOPA uptake in both cell types, was significantly greater than that observed in the presence of extracellular Na+. It is concluded that enhanced ability of OK cells overexpressing Na+-K+-ATPase to translocate Na+ from the apical to the basal cell side correlates positively with their ability to accumulate L-DOPA, which is in agreement with the role of Na+ in taking up the precursor of renal dopamine.
本研究评估了在培养的肾上皮管状细胞中，矢量性Na⁺转运增加是否会促进Na⁺依赖性L-多巴摄取这一假设。在用啮齿动物Na⁺-K⁺-ATP酶α1亚基转染野生型负鼠肾（OK）细胞后，构建出过度表达Na⁺-K⁺-ATP酶的OK细胞，从而实现矢量性Na⁺转运增加。野生型和转染后的OK细胞之间最显著的差异在于，后者过度表达Na⁺-K⁺-ATP酶，且转运体活性增加。对L-多巴摄取饱和曲线的非线性分析显示，野生型和转染细胞中L-多巴摄取的Vmax值（以nmol mg蛋白质/6分钟计）分别为62和80。在OK-WT细胞中，非饱和浓度（0.25μM）的[¹⁴C]-L-多巴摄取不受Na⁺去除的影响，而在OK-α1细胞中，[¹⁴C]-L-多巴的积累明显依赖于细胞外Na⁺的存在。当用胆碱替代Na⁺时，两种细胞类型中中性L-氨基酸对[¹⁴C]-L-多巴摄取的抑制作用（而非碱性和酸性氨基酸的抑制作用）显著大于在细胞外Na⁺存在时观察到的抑制作用。得出的结论是，过度表达Na⁺-K⁺-ATP酶的OK细胞将Na⁺从细胞顶端转运至基底侧的能力增强，这与其积累L-多巴的能力呈正相关，这与Na⁺在摄取肾多巴胺前体中的作用一致。
